Identification of the yeast MCM3-related protein as a component of xenopus DNA replication licensing factor

Kubota, Yumiko; Mimura, Satoru; Nishimoto, Shinichi; Takisawa, Haruhiko; Nishimura, Hiroshi

doi:10.1016/0092-8674(95)90081-0

Cited by 278 publications

(310 citation statements)

References 27 publications

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“…N-terminal-polyhistidine-tagged Xenopus MCM2 was purified as described elsewhere (51). Affinity purification of Xenopus MCM2-7 proteins from the egg extracts was carried out using anti-MCM2 antibodies, essentially as described previously (52,53). In brief, anti-MCM2 antibodies coupled to Protein A-Sepharose beads were incubated with the egg extracts (LSS) for 1 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts

Waga

Zembutsu

2006

Journal of Biological Chemistry

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We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23°C, with one or a few origin recognition complex (ORC) molecules and ϳ10 -20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with ϳ10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions ϳ10 -20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0°C) without geminin, with ϳ10 -20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23°C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas ϳ10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2-7 complexes during metazoan pre-RC formation.DNA replication in eukaryotes is initiated through the coordinated actions of replication initiation proteins. Previous studies have identified the proteins that are involved in the initiation step and an outline of the mechanism of DNA replication initiation in eukaryotic cells has been described (1-3). In brief, the origin recognition complex (ORC), 2 which consists of six subunits (ORC1-6), binds to DNA and then, in cooperation with CDC6 and CDT1, loads the mini-chromosome maintenance (MCM) 2-7 protein complex onto DNA to form a specific protein-DNA complex; the so-called pre-replicative complex (pre-RC). The pre-RC is formed in a cell cycle-regulated manner that involves geminin, a cell cycle-regulated protein that acts as an inhibitor of MCM loading (2, 4). Subsequent activation of the pre-RC is required for unwinding of the DNA around the origin and the start of DNA synthesis (1, 5-11).ORC and most other initiation proteins are well conserved in various eukaryotic species (1). Saccharomyces cerevisiae ORC binds specifically to the autonomously replicating sequence, a DNA sequence functioning as a replicator in DNA replication (12). Fission yeast Schizosaccharomyces pombe ORC contains AT-hook motifs within the ORC4 subunit and thus binds preferentially to A/T-rich sequences that are common in S. pombe origins (13). In contrast, ORC in metazoans such as humans, Drosophila, and Xenopus does not exhibit sequence specificity in its DNA binding (14 -18), but DNA topology has been shown to affect Droso...

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Section: Methodsmentioning

confidence: 99%

Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts

Waga

Zembutsu

2006

Journal of Biological Chemistry

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“…We have previously reported that glutathione S-transferase (GST)-fused recombinant Xenopus MCM3 (maternal form) fails to be transported into nuclei formed in Xenopus egg extracts. 21 To compare the NLS activity of maternal and zygotic forms of MCM3, we examined the nuclear transport of GST-enhanced green fluorescent protein (EGFP) fused recombinant proteins into nuclei formed in the extracts (Fig. 2B).…”

Section: Characterization Of the Nls-like Motifs Found In Maternal Anmentioning

confidence: 99%

Evolutionary diversification ofMCM3genes inXenopus laevisandDanio rerio

et al. 2014

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Embryonic cell cycles of amphibians are rapid and lack zygotic transcription and checkpoint control. At the midblastula transition, zygotic transcription is initiated and cell divisions become asynchronous. Several cell cycle-related amphibian genes retain 2 distinct forms, maternal and zygotic, but little is known about the functional differences between these 2 forms of proteins. The minichromosome maintenance (MCM) 2-7 complex, consisting of 6 MCM proteins, plays a central role in the regulation of eukaryotic DNA replication. Almost all eukaryotes retain just a single MCM gene for each subunit. Here we report that Xenopus and zebrafish have 2 copies of MCM3 genes, one of which shows a maternal and the other a zygotic expression pattern. Phylogenetic analysis shows that the Xenopus and zebrafish zygotic MCM3 genes are more similar to their mammalian MCM3 ortholog, suggesting that maternal MCM3 was lost during evolution in most vertebrate lineages. Maternal MCM3 proteins in these 2 species are functionally different from zygotic MCM3 proteins because zygotic, but not maternal, MCM3 possesses an active nuclear localization signal in its C-terminal region, such as mammalian MCM3 orthologs do. mRNA injection experiments in zebrafish embryos show that overexpression of maternal MCM3 impairs proliferation and causes developmental defects, whereas zygotic MCM3 has a much weaker effect. This difference is brought about by the difference in their C-terminal regions, which contain putative nuclear localization signals; swapping the C-terminal region between maternal and zygotic genes diminishes the developmental defects. This study suggests that evolutionary diversification has occurred in MCM3 genes, leading to distinct functions, possibly as an adaption to the rapid DNA replication required for early development of Xenopus and zebrafish.

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“…MCM homologues have been identified in all eukaryotes, and all are essential for DNA replication (1,(3)(4)(5)(6)(7). The recruitment of these proteins onto replication origins during the G 1 phase of the cell cycle is essential for the formation of a prereplicative complex and initiation of DNA synthesis (8 -12).…”

mentioning

confidence: 99%

The Zinc Finger Domain of the Archaeal Minichromosome Maintenance Protein Is Required for Helicase Activity

Poplawski¹,

Grabowski²,

Long³

et al. 2001

Journal of Biological Chemistry

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The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum ⌬H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX 2 CX n CX 2 C (C 4 ) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif. The minichromosome maintenance (MCM)1 genes encode a family of six proteins (MCM2 to -7) first identified by their essential role in the maintenance of ARS-containing minichromosomes in Saccharomyces cerevisiae (1, 2). MCM homologues have been identified in all eukaryotes, and all are essential for DNA replication (1, 3-7). The recruitment of these proteins onto replication origins during the G 1 phase of the cell cycle is essential for the formation of a prereplicative complex and initiation of DNA synthesis (8 -12). In addition to the heterohexameric structure MCM2/3/4/5/6/7, found in vivo and in vitro, several additional MCM complexes have been identified (13-16). Biochemical studies in yeast and mammals have shown that a dimeric complex of the MCM4/6/7 heterotrimer contains 3Ј-5Ј DNA helicase activity, single-stranded DNA (ssDNA) binding, and DNA-dependent ATPase activities (15,17,18). Both MCM2 and MCM3/5 were shown to inhibit the helicase activity of MCM4/6/7 and thus were suggested to play regulatory roles (15,19). Both genetic and biochemical data suggest that the MCM4/6/7 complex is the eukaryotic replicative helicase and that the other MCM polypeptides may play regulatory roles (15,16,19).Zinc fingers were first identified as zinc-binding domains important for protein-DNA interactions (20, 21). They contain conserved Cys and His residues, and to date more than 10 classes have been biochemically characterized, and their structures have been determined (reviewed in Refs. 22-25). Since their discovery, zinc finger domains have been implicated in diverse functions, including ssDNA and double-stranded DNA (dsDNA) binding, RNA binding, and protein-protein interactions (reviewed in Refs. 24 and 26). The primary amino acid sequences of the eukaryotic MCM2, -4, -6, and -7 proteins from all organisms contain a putative zinc finger motif of the C 4 type, either CX 2 CX n CX 2 C (MCM2) or CX 2 CX n CX 4 C (MCM4, -6, an...

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Identification of the yeast MCM3-related protein as a component of xenopus DNA replication licensing factor

Cited by 278 publications

References 27 publications

Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts

Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts

Evolutionary diversification ofMCM3genes inXenopus laevisandDanio rerio

The Zinc Finger Domain of the Archaeal Minichromosome Maintenance Protein Is Required for Helicase Activity

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